B7-H5, A Costimulatory Polypeptide

ABSTRACT

B7-H5 costimulatory polypeptides, nucleic acids encoding such polypeptides, and methods for using the polypeptides and nucleic acids to enhance a T cell response are provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application Ser. No. 14/645,070, filedMar. 11, 2015, which is a divisional of U.S. Ser. No. 13/852,888, filedMar. 28, 2013 (now U.S. Pat. No. 9,012,409), which is a divisional ofU.S. Ser. No. 13/036,893, filed Feb. 28, 2011 (now U.S. Pat. No.8,426,563), which is a divisional application of U.S. Ser. No.11/571,195, filed Jan. 24, 2008 (now U.S. Pat. No. 7,919,585), which isNational Stage under 35 U.S.C. § 371 of International ApplicationPCT/US2005/022321, filed Jun. 24, 2005, which claims the benefit of thefiling date of U.S. Provisional Patent Application Ser. No. 60/582,491,filed on Jun. 24, 2004. The contents of the prior application are herebyincorporated by reference in their entirety.

BACKGROUND

Activated T lymphocytes play critical roles in host defense againstviral infection and cancer and are involved in the progression ofautoimmune diseases. Two distinct signals are typically required foroptimal activation of antigen-specific T lymphocytes. The first signalis provided by the interactions of the complex of antigenic peptide andmajor histocompatibility complex (MHC) with the T cell receptor (TCR).The second signal is delivered to T cells by costimulatory moleculesexpressed on antigen-presenting cells (APCs). Studies have demonstratedthat costimulatory interactions stimulate T cell growth, up-regulatecytokine production, and promote T cell differentiation. Furthermore,ligation of costimulatory molecules provides a survival signal essentialfor T cells to prevent apoptosis. In addition, costimulatory signalsappear to be critical for the induction and maintenance of T cellanergy. More importantly, there is clear interest in therapeuticmanipulation of costimulatory pathways because the control ofcostimulatory signals can provide a means either to enhance or toinhibit immune responses.

SUMMARY

The invention is based, in part, on the cloning of human and mouse cDNAmolecules encoding novel homologous molecules that co-stimulate the Tcell responses of both species and on the functional characterization ofthe polypeptides that the cDNA molecules encode. The human polypeptideis designated hB7-H5 and the mouse polypeptide mB7-H5. Text that refersto B7-H5 without specifying human versus mouse is pertinent to bothforms of B7-H5. The invention features DNA molecules encoding thehB7-H5, mB7-H5 polypeptides, functional fragments of the polypeptides,and fusion proteins containing the polypeptides or functional fragmentsof the polypeptides, hB7-H5 and mB7-H5 and functional fragments of both,vectors containing the DNA molecules, and cells containing the vectors.Also included in the invention are antibodies that bind to the B7-H5polypeptides. The invention features in vitro, in vivo, and ex vivomethods of co-stimulating T cell responses, methods of screening forcompounds that inhibit or enhance T cell responses, and methods forproducing the above polypeptides and fusion proteins.

Specifically the invention features an isolated nucleic acid (e.g., aDNA) including: (a) a nucleic acid sequence that (i) encodes a B7-H5polypeptide with the ability to co-stimulate a T cell, and (ii)hybridizes under highly stringent conditions to the complement of SEQ IDNO:2 or SEQ ID NO:4 or to the complement of a sequence that encodes apolypeptide with an amino acid sequence with SEQ ID NO:1 or SEQ ID NO:3;or (b) a complement of this nucleic acid sequence. The nucleic acidsequence included in the isolated DNA will be at least 10 bp, 15 bp, 25bp, 50 bp, 75 bp, 100 bp, 125 bp, 150 bp, 175 bp, 200 bp, 250 bp, 300bp, 350 bp, 400 bp, 450 bp, 500 bp, 550 bp, 600 bp, 650 bp, 700 bp, 750,bp 800 bp, 850 bp, 900 bp, 910 bp, 915 bp, 920 bp, 925 bp, 926 bp, 927bp, 928 bp, 929 bp, 930 bp, 931 bp, 932 bp, or 933 bp long. The nucleicacid sequence can encode a B7-H5 polypeptide that includes an aminosequence with SEQ ID NO:1 or SEQ 1D NO:3 with up to 20 (e.g., 0, 2, 5,10, or 15) amino acid insertions, deletions, or substitutions, or it canhave a nucleotide sequence that includes SEQ ID NO:2 or SEQ ID NO:4. Thenucleic acid sequence can also encode functional fragments of theseB7-H5 polypeptides, e.g., fragments with the ability to co-stimulate a Tcell.

The invention also embodies an isolated B7-H5 polypeptide, e.g., apolypeptide encoded by a DNA that includes a nucleic acid sequence that(i) encodes a polypeptide with the ability to co-stimulate a T cell and(ii) hybridizes under stringent conditions to the complement of asequence that encodes a polypeptide with an amino acid sequence with SEQID NO:1 or SEQ ID NO:3. The B7-H5 polypeptide can include an aminosequence of amino acid residue 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 to amino acid residue 311of SEQ ID NO:1 or amino acid residue 309 of SEQ ID NO:3. The inventionalso encompasses B7-H5 polypeptides that include an amino acid sequencewith SEQ ID NO:1 or SEQ ID NO:3, or either of these amino acid sequencesbut differing solely by not more than 20 (e.g., not more than 15, 10, 5,2, or 1) conservative substitutions or by not more than 20 (e.g., notmore than 15, 10, 5, 2, or 1) amino acid insertions, deletions, orsubstitutions, and further encompasses functional fragments of any ofthese polypeptides, e.g., a fragment that has the ability toco-stimulate a T cell. Also included are isolated immunogenicpolypeptides comprising at least 8 (e.g., 10, 15, 20, 25, 30, 40, or 50)consecutive residues of the polypeptides disclosed herein. Thepolypeptides of the invention include fusion proteins containing a firstdomain and at least one additional domain. The first domain can be anyof the B7-H5 polypeptides described above or a functional fragment ofany of these polypeptides. The at least one additional domain can be,for example, a heterologous targeting or leader sequence, or an aminoacid sequence that facilitates purification, detection, or solubility ofthe fusion protein. The second domain can be, for example, all or partof an immunoglobulin (Ig) heavy chain constant region. Also included areisolated nucleic acid molecules encoding the fusion proteins.

The invention features vectors containing any of the nucleic acids ofthe invention and nucleic acid molecules encoding the fusion proteins ofthe invention. The vectors can be expression vectors in which thenucleic acid coding sequence or molecule is operably linked to anexpression control sequence, e.g., a regulatory element that allowsexpression of the nucleic acid sequence or molecule in a cell. Alsoincluded in the invention are cells (e.g., mammalian, insect, yeast,fungal, or bacterial cells) containing any of the vectors of theinvention.

Another embodiment of the invention is a method of co-stimulating a Tcell that involves contacting the T cell with any of the B7-H5polypeptides of the invention, functional fragments thereof, or fusionproteins of the invention; these 3 classes of molecule are, forconvenience, designated “B7-H5 agents.” The contacting can be byculturing any of these B7-H5 agents with the T cell in vitro.Alternatively, the T cell can be in a mammal (e.g., a human, non-humanprimate (e.g., monkey), mouse, rat, guinea pig, cow, sheep, horse, pig,rabbit, dog, or cat) and the contacting can be, for example, byadministering any of the B7-H5 agents to the mammal or administering anucleic acid encoding the B7-H5 agent to the mammal. In addition, themethod can be an ex vivo procedure that involves providing a recombinantcell which is the progeny of a cell obtained from the mammal and hasbeen transfected or transformed ex vivo with a nucleic acid encoding anyof the B7-H5 agents so that the cell expresses the B7-H5 agent; andadministering the cell to the mammal. In this ex vivo procedure, thecell can be an antigen presenting cell (APC) that expresses the B7-H5agent on its surface. Furthermore, prior to administering to the mammal,the APC can be pulsed with an antigen or an antigenic peptide. In any ofthe above methods, the mammal can be suspected of having, for example,an immunodeficiency disease, an inflammatory condition, or an autoimmunedisease. In addition, in any of the methods, the T cell can be a helperT cell, e.g., a T cell that helps an effector (e.g., a cytotoxic Tlymphocyte (CTL) or B cell antibody) response. An antibody response canbe, for example, an IgM, IgG1, IgG2a, IgG2b, IgG3, IgG4, IgE, or IgAantibody response. Co-stimulation of a T cell by any of the B7-H5 agentscan result in an increase in the level of CD40 ligand on the surface ofthe T cell.

The invention includes a method of identifying a compound that inhibitsan immune response. The method involves: providing a test compound;culturing, together, the compound, one or more B7-H5 agents, a T cell,and a T cell activating stimulus; and determining whether the testcompound inhibits the response of the T cell to the stimulus, as anindication that the test compound inhibits an immune response. Theinvention also embodies a method of identifying a compound that enhancesan immune response. The method involves: providing a test compound;culturing, together, the compound, one or more of B7-H5 agents, a Tcell, and a T cell activating stimulus; and determining whether the testcompound enhances the response of the T cell to the stimulus, as anindication that the test compound enhances an immune response. In boththese methods, the stimulus can be, for example, an antibody that bindsto a T cell receptor or a CD3 polypeptide. Alternatively, the stimuluscan be an alloantigen or an antigenic peptide bound to a majorhistocompatibility complex (MHC) molecule on the surface of an antigenpresenting cell (APC). The APC can be transfected or transformed with anucleic acid encoding the B7-H5 agent and the B7-H5 agent can beexpressed on the surface of the APC.

The invention also features an antibody (e.g., a polyclonal or amonoclonal antibody) that binds to any of the B7-H5 polypeptides of theinvention, e.g., the polypeptide with SEQ ID NO:1 or SEQ ID NO:3, e.g.,the 5H9 or the 1H11 monoclonal antibody disclosed herein. The inventionalso provides hybridomas secreting such monoclonal antibodies.

The invention also features a method of producing any of the B7-H5polypeptides of the invention, functional fragments thereof, or fusionproteins of the invention. The method involves culturing a cell of theinvention and purifying the relevant B7-H5 protein from the culture.

“Polypeptide” and “protein” are used interchangeably and mean anypeptide-linked chain of amino acids, regardless of length orpost-translational modification. The invention also features B7-H5polypeptides with conservative substitutions. Conservative substitutionstypically include substitutions within the following groups: glycine andalanine; valine, isoleucine, and leucine; aspartic acid and glutamicacid; asparagine, glutamine, serine and threonine; lysine, histidine andarginine; and phenylalanine and tyrosine.

The term “isolated” polypeptide or peptide fragment as used hereinrefers to a polypeptide or a peptide fragment which either has nonaturally-occurring counterpart (e.g., a peptidomimetic), or has beenseparated or purified from components which naturally accompany it,e.g., in tissues such as pancreas, liver, spleen, ovary, testis, muscle,joint tissue, neural tissue, gastrointestinal tissue, or body fluidssuch as blood, serum, or urine. Typically, the polypeptide or peptidefragment is considered “isolated” when it is at least 70%, by dryweight, free from the proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, a preparation of apolypeptide (or peptide fragment thereof) of the invention is at least80%, more preferably at least 90%, and most preferably at least 99%, bydry weight, the polypeptide (or the peptide fragment thereof),respectively, of the invention. Thus, for example, a preparation ofpolypeptide x is at least 80%, more preferably at least 90%, and mostpreferably at least 99%, by dry weight, polypeptide x. Since apolypeptide that is chemically synthesized is, by its nature, separatedfrom the components that naturally accompany it, the syntheticpolypeptide or nucleic acid is “isolated.”

An isolated polypeptide (or peptide fragment) of the invention can beobtained, for example, by extraction from a natural source (e.g., fromhuman tissues or bodily fluids); by expression of a recombinant nucleicacid encoding the peptide; or by chemical synthesis. A peptide that isproduced in a cellular system different from the source from which itnaturally originates is “isolated,” because it will be separated fromcomponents which naturally accompany it. The extent of isolation orpurity can be measured by any appropriate method, e.g., columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

An “isolated DNA” means DNA free of one or both of the genes that flankthe gene containing the DNA of interest in the genome of the organism inwhich the gene containing the DNA of interest naturally occurs. The termtherefore includes a recombinant DNA incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote. It also includes a separate molecule such as: acDNA where the corresponding genomic DNA has introns and therefore adifferent sequence; a genomic fragment; a fragment produced bypolymerase chain reaction (PCR); a restriction fragment; a DNA encodinga non-naturally occurring protein, fusion protein, or fragment of agiven protein; or a nucleic acid which is a degenerate variant of anaturally occurring nucleic acid. In addition, it includes a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein. Also included is a recombinant DNA that includes aportion of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. Itwill be apparent from the foregoing that isolated DNA does not mean aDNA present among hundreds to millions of other DNA molecules within,for example, cDNA or genomic DNA libraries or genomic DNA restrictiondigests in, for example, a restriction digest reaction mixture or anelectrophoretic gel slice.

As used herein, a polypeptide that “co-stimulates” a T cell is apolypeptide that, upon interaction with a cell-surface molecule on the Tcell, enhances the response of the T cell. The T cell response thatresults from the interaction will be greater than the response in theabsence of the polypeptide. The response of the T cell in the absence ofthe co-stimulatory polypeptide can be no response or it can be aresponse significantly lower than in the presence of the co-stimulatorypolypeptide. It is understood that the response of the T cell can be aneffector (e.g., CTL or antibody-producing B cell) response, a helperresponse providing help for one or more effector (e.g., CTL orantibody-producing B cell) responses, or a suppressive response.

As used herein, an “activating stimulus” is a molecule that delivers anactivating signal to a T cell, preferably through the antigen specific Tcell receptor (TCR). The activating stimulus can be sufficient to elicita detectable response in the T cell. Alternatively, the T cell mayrequire co-stimulation (e.g., by a B7-H5 polypeptide) in order torespond detectably to the activating stimulus. Examples of activatingstimuli include, without limitation, antibodies that bind to the TCR orto a polypeptide of the CD3 complex that is physically associated withthe TCR on the T cell surface, alloantigens, or an antigenic peptidebound to a MHC molecule.

As used herein, a “fragment” of a B7-H5 polypeptide is a fragment of thepolypeptide that is shorter than the full-length, immature polypeptide.Generally, fragments will be 5 or more amino acids, e.g., 6, 7, 8, 9,10, 12, 15, 18, 21, 25, 30, 35, 40, 50, 60, 80, 100, 120, 150, 180, 210,240, 260, 270, 280, 285, 290, 295, 300, 303, 306, or 308 or more aminoacids, in length. An antigenic fragment has the ability to be recognizedand bound by an antibody. In certain embodiments, antigenic fragmentsare also functional fragments.

As used herein, a “functional fragment” of a B7-H5 polypeptide is afragment of the polypeptide that is shorter than the full-length,immature polypeptide and has at least 25% (e.g., at least 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 98%, 99%, or even 100% or more) of the abilityof the full-length mature B7-H5 polypeptide to co-stimulate a T cell.Methods of establishing whether a fragment of an B7-H5 molecule isfunctional are known in the art. For example, fragments of interest canbe made by either recombinant, synthetic, or proteolytic digestivemethods. Such fragments can then be isolated and tested for theirability to co-stimulate T cells by procedures described herein.

As used herein, “operably linked” means incorporated into a geneticconstruct so that expression control sequences effectively controlexpression of a coding sequence of interest.

As used herein, the term “antibody” refers not only to whole antibodymolecules, but also to antigen-binding fragments, e.g., Fab, F(ab′)2,Fv, and single chain Fv fragments. Also included are chimericantibodies.

As used herein, an antibody that “binds specifically” to an isolatedB7-H5 polypeptide encoded by a DNA that includes a nucleic acid sequencethat (i) encodes a polypeptide with the ability to co-stimulate a T celland (ii) hybridizes under stringent conditions to the complement of asequence that encodes a polypeptide with an amino acid sequence with SEQID NO:1 or SEQ ID NO:3, is an antibody that does not bind to B7-1, B7-2,B7-H1, B7-H2, B7-H3, or B7H-4 polypeptides.

Unless otherwise defined, all technical and, scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. In case of conflict,the present document, including definitions, will control. Preferredmethods and materials are described below, although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention. All publications,patent applications, patents and other references mentioned herein areincorporated by reference in their entirety. The materials, methods, andexamples disclosed herein are illustrative only and not intended to belimiting.

Other features and advantages of the invention, e.g., enhancing immuneresponses in mammalian subjects, will be apparent from the followingdescription, from the drawings and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a nucleotide sequence (SEQ ID NO:5) of a cDNA clone thatincludes a sequence encoding a human B7-H5 polypeptide. The B7-H5 startcodon is indicated by bold and underscore. The B7-H5 stop codon isindicated by underscore.

FIG. 2 is a nucleotide sequence (SEQ ID NO:6) that includes a sequenceencoding a mouse B7-H5 polypeptide. The B7-H5 start codon is indicatedby bold and underscore. The B7-H5 stop codon is indicated by underscore.

FIG. 3 is an annotated amino acid sequence (SEQ ID NO:1) of human B7-H5.The IgV-like domain is underlined; a predicted intermolecular cysteineis indicated in bold and underscore; the predicted transmembrane domainis indicated in bold; a tyrosine that is potentially subject tophosphorylation is boxed.

FIG. 4 is an annotated amino acid sequence (SEQ ID NO:3) of mouse B7-H5.The IgV-like domain is underlined; a predicted intermolecular cysteineis indicated by bold and underscore; the predicted transmembrane domainis indicated in bold; tyrosines that are potentially subject tophosphorylation are boxed.

FIG. 5 is a representation of the domain structure of human B7-H5. Apredicted signal sequence includes residues 1-29 of SEQ ID NO:1. Apredicted immunoglobulin-like (Ig) domain includes residues 30-170 ofSEQ ID NO:1. A predicted linker domain includes residues 171-194 of SEQID NO:1. A predicted transmembrane domain includes residues 195-216 ofSEQ ID NO:1. Residues 217-311 form a predicted intracellular domain,with residues 280-292 forming a low complexity/structured motif. Thedomain structure and topology are conserved between the human and mouseB7-H5 polypeptides.

FIG. 6 is a structure-based alignment of B7-H5 and other B7 familymembers. Aligned are the amino acid sequences of segments of the B7family (h: human, m: mouse) polypeptides (including B7-H5) that containimmunoglobulin superfamily (IgSF) V-domain sequences. IgSF V-setconsensus residues are shown on a black background. Consensus residuepositions are labeled with invariant IgSF residues or conserved residuecharacter (h: hydrophobic, p: polar). Residue positions shown on a graybackground are signature residues of the B7 family outside IgSFconsensus positions. Beta-strands of the V-domain are designatedaccording to IgSF conventions (A′, B, C, C′, C″, D, F, G). Residuepositions labeled with # are involved in dimerization of CD80/CD86 intheir crystal structures and residues labeled with asterisks participatein CTLA4 binding. The amino acid sequences of the hCD80, hCD86, hB7-H1,mB7-H1, hB7-H2, hB7-H3, hB7-DC, mB7-DC, hB7-H4, mB7-H4, hB7-H5, andmB7-H5 segments are set forth herein as SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22,respectively.

FIG. 7 is a line graph depicting in vitro co-stimulation of T cellproliferation by B7-H5. T cell proliferation is depicted as ³H-thymidineincorporation (y-axis; counts per minute, cpm). Anti-CD3 antibodyconcentration is indicated on the x-axis.

FIG. 8A is a fluorescence flow cytometry (FFC) histogram depictingnumber of cells (y-axis) with the indicated fluorescence levels(x-axis). CHO cell transfectants expressing human B7-H5 (open) ormock-transfected CHO cells (solid) were stained with the 5H9 monoclonalantibody.

FIG. 8B is a FFC histogram depicting number of cells (y-axis) with theindicated fluorescence levels (x-axis). CHO cell transfectantsexpressing human B7-H5 (open) or mock-transfected CHO cells (solid) werestained with the 1H11 monoclonal antibody.

FIG. 9 is a translated nucleotide sequence (SEQ ID NO:2) of a cDNAencoding human B7-H5.

FIG. 10 is a translated nucleotide sequence (SEQ ID NO:4) of a cDNAencoding mouse B7-H5.

DETAILED DESCRIPTION

The applicants have discovered, inter alia, a new member of the B7family of costimulatory molecules, which has been designated B7-H5. Boththe human and mouse B7-H5 polypeptides and nucleotides encoding them aredisclosed. Similar to other members of the B7 family, these B7-H5polypeptides can co-stimulate T cells.

Nucleic Acid Molecules

The B7-H5 nucleic acid molecules of the invention can be cDNA, genomicDNA, synthetic DNA, or RNA, and can be double-stranded orsingle-stranded (i.e., either a sense or an antisense strand or both).Fragments of these molecules are also considered within the scope of theinvention, and can be produced by, for example, the polymerase chainreaction (PCR) or generated by treatment with one or more restrictionendonucleases. A ribonucleic acid (RNA) molecule can be produced by invitro transcription. Preferably, the nucleic acid molecules encodepolypeptides that, regardless of length, are soluble under normalphysiological conditions.

The nucleic acid molecules of the invention can contain naturallyoccurring sequences, or sequences that differ from those that occurnaturally, but, due to the degeneracy of the genetic code, encode thesame polypeptide (for example, the polypeptides with SEQ ID NOs:1 or 3).In addition, these nucleic acid molecules are not limited to codingsequences, e.g., they can include some or all of the non-codingsequences that lie upstream or downstream from a coding sequence.

Nucleic acids of the invention can be nucleic acid analogs. Nucleic acidanalogs can be modified at the base moiety, sugar moiety, or phosphatebackbone. Such modification can improve, for example, stability,hybridization, or solubility of the nucleic acid. Modifications at thebase moiety can include deoxyuridine for deoxythymidine, and5-methyl-2′-deoxycytidine or 5-bromo-2′-deoxycytidine for deoxycytidine.Modifications of the sugar moiety can include modification of the 2′hydroxyl of the ribose sugar to form 2′-O-methyl or 2′-O-allyl sugars.The deoxyribose phosphate backbone can be modified to produce morpholinonucleic acids, in which each base moiety is linked to a six membered,morpholino ring, or peptide nucleic acids, in which the deoxyphosphatebackbone is replaced by a pseudopeptide backbone and the four bases areretained. See, for example, Summerton and Weller (1997) AntisenseNucleic Acid Drug Dev. 7:187-195; and Hyrup et al. (1996) Bioorgan. Med.Chem. 4:5-23. In addition, the deoxyphosphate backbone can be replacedwith, for example, a phosphorothioate or phosphorodithioate backbone, aphosphoroamidite, or an alkyl phosphotriester backbone.

The nucleic acid molecules of the invention can be synthesized (forexample, by phosphoramidite-based synthesis) or obtained from abiological cell, such as the cell of a mammal. Thus, the nucleic acidscan be those of a human, non-human primate (e.g., monkey) mouse, rat,guinea pig, cow, sheep, horse, pig, rabbit, dog, or cat.

In addition, the isolated nucleic acid molecules of the inventionencompass segments that are not found as such in the natural state.Thus, the invention encompasses recombinant nucleic acid molecules (forexample, isolated nucleic acid molecules encoding B7-H5) incorporatedinto a vector (for example, a plasmid or viral vector) or into thegenome of a heterologous cell or into the genome of a homologous cell ata position other than the natural chromosomal location. Recombinantnucleic acid molecules and uses therefor are discussed further below.

Certain nucleic acid molecules of the invention are antisense moleculesor are transcribed into antisense molecules. These can be used, forexample, to down-regulate translation of B7-H5 mRNA within a cell.Techniques associated with detection or regulation of genes are wellknown to skilled artisans and such techniques can be used to diagnoseand/or treat disorders associated with aberrant B7-H5 expression.Nucleic acid molecules of the invention are discussed further below inthe context of their therapeutic utility.

A B7-H5 family gene or protein can be identified based on its similarityto the relevant B7-H5 gene or protein, respectively. For example, theidentification can be based on sequence identity. The invention featuresisolated nucleic acid molecules which are identical to, or are at least50% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to: (a) a nucleicacid molecule that encodes the polypeptide of SEQ ID NO:1 or SEQ IDNO:3; (b) the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4; or (c)a nucleic acid molecule which includes a segment of at least 10 (e.g.,at least 15, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 910, 915, 920, 925,926, 927, 928, 929, 930, 931, 932, or 933) nucleotides of SEQ ID NO:2 orSEQ ID NO:4.

The determination of percent identity between two sequences isaccomplished using the mathematical algorithm of Karlin and Altschul,Proc. Natl. Acad. Sci. USA 90, 5873-5877, 1993. Such an algorithm isincorporated into the BLASTN and

BLASTP programs of Altschul et al. (1990) J Mol. Biol. 215, 403-410.BLAST nucleotide searches are performed with the BLASTN program,score=100, wordlength=12 to obtain nucleotide sequences homologous toB7-H5-encoding nucleic acids. BLAST protein searches are performed withthe BLASTP program, score=50, wordlength=3 to obtain amino acidsequences homologous to B7-H5. To obtain gapped alignments forcomparative purposes, Gapped BLAST is utilized as described in Altschulet al. (1997) Nucleic Acids Res. 25, 3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) are used (see ncbi.nlm.nih.gov).

Hybridization can also be used as a measure of homology between twonucleic acid sequences. A B7-H5-encoding nucleic acid sequence, or aportion thereof, can be used as hybridization probe according tostandard hybridization techniques. The hybridization of a B7-H5 probe toDNA from a test source (e.g., a mammalian cell) is an indication of thepresence of B7-H5 DNA in the test source. Hybridization conditions areknown to those skilled in the art and can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6, 1991.Moderate hybridization conditions are defined as equivalent tohybridization in 2× sodium chloride/sodium citrate (SSC) at 30° C.,followed by one or more washes in 1× SSC, 0.1% SDS at 50-60° C. Highlystringent conditions are defined as equivalent to hybridization in 6xsodium chloride/sodium citrate (SSC) at 45° C., followed by one or morewashes in 0.2x SSC, 0.1% SDS at 50-65° C.

The invention also encompasses: (a) vectors that contain any of theforegoing B7-H5- related coding sequences and/or their complements (thatis, “antisense” sequence); (b) expression vectors that contain any ofthe foregoing B7-H5-related coding sequences operatively linked to oneor more transcriptional/translational regulatory elements (examples ofwhich are given below) that direct expression of the coding sequences;(c) expression vectors containing, in addition to sequences encoding aB7-H5 polypeptide, nucleic acid sequences that are unrelated to nucleicacid sequences encoding B7-H5, such as molecules encoding a reporter,marker, or a signal peptide, e.g., fused to B7-H5; and (d) geneticallyengineered host cells that contain any of the foregoing expressionvectors and thereby express the nucleic acid molecules of the invention.

Recombinant nucleic acid molecules can contain a sequence encoding B7-H5having a heterologous signal sequence. The full length B7-H5polypeptide, a domain of B7-H5, or a fragment thereof may be fused toadditional polypeptides, as described below. Similarly, the nucleic acidmolecules of the invention can encode the mature form of B7-H5 or a formthat includes an exogenous polypeptide that facilitates secretion.

The transcriptional/translational regulatory elements referred to aboveand which are further described below, include, but are not limited to,inducible and non-inducible promoters, enhancers, operators and otherelements, which are known to those skilled in the art, and which driveor otherwise regulate gene expression. Such regulatory elements includebut are not limited to the cytomegalovirus hCMV immediate early gene,the early or late promoters of SV40 adenovirus, the lac system, the trpsystem, the TAC system, the TRC system, the major operator and promoterregions of phage A, the control regions of fd coat protein, the promoterfor 3-phosphoglycerate kinase, the promoters of acid phosphatase, andthe promoters of the yeast α-mating factors.

Similarly, the nucleic acid can form part of a hybrid gene encodingadditional polypeptide sequences, for example, sequences that functionas a marker or reporter. Examples of marker or reporter genes includeβ-lactamase, chloramphenicol acetyltransferase (CAT), adenosinedeaminase (ADA), aminoglycoside phosphotransferase (neo^(r), G418^(r)),dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH),thymidine kinase (TK), lacZ (encoding β-galactosidase), and xanthineguanine phosphoribosyltransferase (XGPRT). As with many of the standardprocedures associated with the practice of the invention, skilledartisans will be aware of additional useful reagents, for example,additional sequences that can serve the function of a marker orreporter. Generally, the hybrid polypeptide will include a first portionand a second portion; the first portion being a B7-H5 polypeptide (orany of the fragment of such a polypeptide disclosed herein) and thesecond portion being, for example, the reporter described above or an Igconstant region or part of an Ig constant region, e.g., the CH2 and CH3domains of IgG2a.

The expression systems that may be used for purposes of the inventioninclude, but are not limited to, microorganisms such as bacteria (forexample, E. coli and B . subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA, or cosmid DNA expression vectorscontaining the nucleic acid molecules of the invention; yeast (forexample, Saccharomyces and Pichia) transformed with recombinant yeastexpression vectors containing the nucleic acid molecules of theinvention, preferably containing a nucleic acid sequence (e.g., SEQ IDNO:2 or 4) encoding a B7-H5 polypeptide; insect cell systems infectedwith recombinant virus expression vectors (for example, baculovirus)containing the nucleic acid molecules of the invention; plant cellsystems infected with recombinant virus expression vectors (for example,cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) ortransformed with recombinant plasmid expression vectors (for example, Tiplasmid) containing B7-H5 nucleotide sequences; or mammalian cellsystems (for example, COS, CHO, BHK, 293, VERO, HeLa, MDCK, WI38, andNIH 3T3 cells) harboring recombinant expression constructs containingpromoters derived from the genome of mammalian cells (for example, themetallothionein promoter) or from mammalian viruses (for example, theadenovirus late promoter and the vaccinia virus 7.5K promoter). Alsouseful as host cells are primary or secondary cells obtained directlyfrom a mammal transfected with a plasmid vector or infected with a viralvector.

A host cell (e.g., a prokaryotic cell or a eukaryotic cell such as a COScell) can be used to, for example, produce the costimulatorypolypeptides provided herein. In some embodiments, a host cell (e.g., anAPC) can be used to express the costimulatory polypeptides of theinvention for presentation to a T cell.

Polypeptides and Polypeptide Fragments

The polypeptides of the invention include B7-H5 and functional fragmentsthereof. The polypeptides disclosed herein also include fusion proteinswhich contain either full-length B7-H5 or a functional fragment thereoffused to an unrelated amino acid sequence. The unrelated sequences canbe additional functional domains or signal peptides. Signal peptides aredescribed in greater detail and exemplified below. The polypeptides canalso be any of those described above but with one or more conservativesubstitutions.

The polypeptides can be purified from natural sources (e.g., blood,serum plasma, tissues, or cells such as T cells or any cell thatnaturally produces B7-H5). Polypeptides can also be convenientlysynthesized by standard chemical means. In addition, polypeptides can beproduced by standard in vitro recombinant DNA techniques and in vivorecombination/genetic recombination (e.g., transgenesis), using thenucleotide sequences encoding the appropriate polypeptides. Methods wellknown to those skilled in the art can be used to construct expressionvectors containing relevant coding sequences and appropriatetranscriptional/translational control signals. See, for example, thetechniques described in Sambrook et al., Molecular Cloning: A LaboratoryManual (2nd Ed.) (Cold Spring Harbor Laboratory, N.Y., 1989), andAusubel et al., Current Protocols in Molecular Biology, (GreenPublishing Associates and Wiley Interscience, N.Y., 1989).

Polypeptides and fragments of the invention also include those describedabove, but modified for in vivo use by the addition, the amino- and/orcarboxyl-terminal ends, of a blocking agent to facilitate survival ofthe relevant polypeptide in vivo. This can be useful in those situationsin which the peptide termini tend to be degraded by proteases prior tocellular uptake. Such blocking agents can include, without limitation,additional related or unrelated peptide sequences that can be attachedto the amino and/or carboxyl terminal residues of the peptide to beadministered. This can be done either chemically during the synthesis ofthe peptide or by recombinant DNA technology by methods familiar toartisans of average skill.

Alternatively, blocking agents such as pyroglutamic acid or othermolecules known in the art can be attached to the amino and/or carboxylterminal residues, or the amino group at the amino terminus or carboxylgroup at the carboxyl terminus can be replaced with a different moiety.Likewise, the peptides can be covalently or noncovalently coupled topharmaceutically acceptable “carrier” proteins prior to administration.

B7-H5 polypeptides can be purified using, for example, chromatographicmethods such as DEAE ion exchange, gel filtration, and hydroxyapatitechromatography. For example, a B7-H5 polypeptide in a cell culturesupernatant or a cytoplasmic extract can be purified using a protein Gcolumn. In some embodiments, B7-H5 polypeptides can be “engineered” tocontain an amino acid sequence that allows the polypeptides to becaptured onto an affinity matrix. For example, a tag such as c-myc,glutathione S-transferase (GST), hemagglutinin, polyhistidine, or Flag™(Kodak) can be used to aid polypeptide purification. Such tags can beinserted anywhere within the polypeptide, including at either thecarboxyl or amino terminus. Immunoaffinity chromatography also can beused to purify costimulatory polypeptides.

Also of interest are peptidomimetic compounds that are designed basedupon the amino acid sequences of the functional peptide fragments.Peptidomimetic compounds are synthetic compounds having athree-dimensional conformation (i.e., a “peptide motif”) that issubstantially the same as the three-dimensional conformation of aselected peptide. The peptide motif provides the peptidomimetic compoundwith the ability to co-stimulate T cells in a manner qualitativelyidentical to that of the B7-H5 functional peptide fragment from whichthe peptidomimetic was derived. Peptidomimetic compounds can haveadditional characteristics that enhance their therapeutic utility, suchas increased cell permeability and prolonged biological half-life.

The peptidomimetics typically have a backbone that is partially orcompletely non-peptide, but with side groups that are identical to theside groups of the amino acid residues that occur in the peptide onwhich the peptidomimetic is based. Several types of chemical bonds,e.g., ester, thioester, thioamide, retroamide, reduced carbonyl,dimethylene and ketomethylene bonds, are known in the art to begenerally useful substitutes for peptide bonds in the construction ofprotease-resistant peptidomimetics.

Methods of Co-Stimulating a T Cell

The methods of the invention involve contacting a T cell with a B7-H5polypeptide of the invention, or a functional fragment thereof, in orderto co-stimulate the T cell. Such polypeptides or functional fragmentscan have amino acid sequences identical to wild-type sequences or theycan contain one or more conservative substitutions. The contacting canoccur before, during, or after activation of the T cell. Contacting ofthe T cell with the B7-H5 polypeptide will preferably be atsubstantially the same time as activation. Activation can be, forexample, by exposing the T cell to an antibody that binds to the T cellreceptor (TCR) or one of the polypeptides of the CD3 complex that isphysically associated with the TCR. Alternatively, the T cell can beexposed to either an alloantigen (e.g., a major histocompatibility (MHC)alloantigen) on, for example, an antigen presenting cell (APC) (e.g., adendritic cell, a macrophage, a monocyte, or a B cell) or an antigenicpeptide produced by processing of a protein antigen by any of the aboveAPC and presented to the T cell by MHC molecules on the surface of theAPC. The T cell can be a CD4⁺ T cell or a CD8⁺ T cell. The B7-H5polypeptide can be added to the solution containing the cells, or it canbe expressed on the surface of an APC, e.g., an APC presenting analloantigen or an antigen peptide bound to an MHC molecule.Alternatively, if the activation is in vitro, the B7-H5 polypeptide canbe bound to a surface of the relevant culture vessel, e.g., a well of aplastic microtiter plate.

The methods can be performed in vitro, in vivo, or ex vivo. In vitroapplication of B7-H5 can be useful, for example, in basic scientificstudies of immune mechanisms or for production of activated T cells foruse in either studies on T cell function or, for example, passiveimmunotherapy. Furthermore, B7-H5 could be added to in vitro assays(e.g., in T cell proliferation assays) designed to test for immunity toan antigen of interest in a subject from which the T cells wereobtained. Addition of B7-H5 to such assays would be expected to resultin a more potent, and therefore more readily detectable, in vitroresponse. However, the methods of the invention will preferably be invivo or ex vivo (see below).

The B7-H5 polypeptides and variants thereof are generally useful asimmune response-stimulating therapeutics. For example, the polypeptidesof the invention can be used for treatment of disease conditionscharacterized by immunosuppression: e.g., cancer, AIDS or AIDS-relatedcomplex, other virally or environmentally-induced conditions, andcertain congenital immune deficiencies. The polypeptides may also beemployed to increase immune function that has been impaired by the useof radiotherapy or immunosuppressive drugs such as certainchemotherapeutic agents, and therefore are particularly useful whengiven in conjunction with such drugs or radiotherapy. The polypeptidescan, furthermore, be used to enhance immune responses in normalsubjects.

These methods of the invention can be applied to a wide range ofspecies, e.g., humans, non-human primates, horses, cattle, pigs, sheep,goats, dogs, cats, rabbits, guinea pigs, hamsters, rats, and mice.

In Vivo Approaches

In one in vivo approach, a B7-H5 polypeptide (or a functional fragmentthereof) itself is administered to the subject. Generally, the compoundsof the invention will be suspended in a pharmaceutically-acceptablecarrier (e.g., physiological saline) and administered orally or byintravenous infusion, or injected subcutaneously, intramuscularly,intraperitoneally, intrarectally, intravaginally, intranasally,intragastrically, intratracheally, or intrapulmonarily. They arepreferably delivered directly to an appropriate lymphoid tissue (e.g.spleen, lymph node, or mucosal-associated lymphoid tissue (MALT)). Thedosage required depends on the choice of the route of administration,the nature of the formulation, the nature of the patient's illness, thesubject's size, weight, surface area, age, and sex, other drugs beingadministered, and the judgment of the attending physician. Suitabledosages are in the range of 0.01-10 mg/kg. Wide variations in the neededdosage are to be expected in view of the variety of polypeptides andfragments available and the differing efficiencies of various routes ofadministration. For example, oral administration would be expected torequire higher dosages than administration by i.v. injection. Variationsin these dosage levels can be adjusted using standard empirical routinesfor optimization as is well understood in the art. Administrations canbe single or multiple (e.g., 2- or 3-, 4-, 6-, 8-, 10-, 20-, 50-,100-,150-, or more fold). Encapsulation of the polypeptide in a suitabledelivery vehicle (e.g., polymeric microparticles or implantable devices)may increase the efficiency of delivery, particularly for oral delivery.

Alternatively, a polynucleotide containing a nucleic acid sequenceencoding a B7-H5 polypeptide or functional fragment thereof can bedelivered to an appropriate cell of the animal. Expression of the codingsequence will preferably be directed to lymphoid tissue of the subjectby, for example, delivery of the polynucleotide to the lymphoid tissue.This can be achieved by, for example, the use of a polymeric,biodegradable microparticle or microcapsule delivery vehicle, sized tooptimize phagocytosis by phagocytic cells such as macrophages. Forexample, PLGA (poly-lacto-co-glycolide) microparticles approximately1-10 μm in diameter can be used. The polynucleotide is encapsulated inthese microparticles, which are taken up by macrophages and graduallybiodegraded within the cell, thereby releasing the polynucleotide. Oncereleased, the DNA is expressed within the cell. A second type ofmicroparticle is intended not to be taken up directly by cells, butrather to serve primarily as a slow-release reservoir of nucleic acidthat is taken up by cells only upon release from the micro-particlethrough biodegradation. These polymeric particles should therefore belarge enough to preclude phagocytosis, i.e., larger than 5 μm andpreferably larger than 20 μm.

Another way to achieve uptake of the nucleic acid is using liposomes,prepared by standard methods. The vectors described herein can beincorporated alone into these delivery vehicles or co-incorporated withtissue-specific antibodies. Alternatively, one can prepare a molecularconjugate composed of a plasmid or other vector attached topoly-L-lysine by electrostatic or covalent forces. Poly-L-lysine bindsto a ligand that can bind to a receptor on target cells (Cristiano etal. (1995), J. Mol. Med. 73, 479). Alternatively, lymphoid tissuespecific targeting can be achieved by the use of lymphoidtissue-specific transcriptional regulatory elements (TRE) such as a Blymphocyte, T lymphocyte, or dendritic cell specific TRE. Lymphoidtissue specific TRE are known (Thompson et al. (1992), Mol. Cell. Biol.12, 1043-1053; Todd et al. (1993), J. Exp. Med. 177, 1663-1674; Penix etal. (1993), J. Exp. Med. 178, 1483-1496). Delivery of “naked DNA” (i.e.,without a delivery vehicle) to an intramuscular, intradermal, orsubcutaneous site, is another means to achieve in vivo expression.

In the relevant polynucleotides (e.g., expression vectors) the nucleicacid sequence encoding a B7-H5 polypeptide or functional fragment ofinterest with an initiator methionine and optionally a targetingsequence is operatively linked to a promoter or enhancer-promotercombination.

Short amino acid sequences can act as signals to direct proteins tospecific intracellular compartments. For example, hydrophobic signalpeptides (e.g., MAISGVPVLGFFIIAVLMSAQESWA (SEQ ID NO:7)) are found atthe amino terminus of proteins destined for the endoplasmic reticulum(ER). Whereas the sequence KFERQ (SEQ ID NO:8) and other closely relatedsequences are known to target intracellular polypeptides to lysosomes,other sequences (e.g., MDDQRDLISNNEQLP (SEQ ID NO:9) direct polypeptidesto endosomes. In addition, the peptide sequence KDEL (SEQ ID NO:10) hasbeen shown to act as a retention signal for the ER. Each of these signalpeptides, or a combination thereof, can be used to traffic the B7-H5polypeptides or functional fragments of the invention as desired. DNAsencoding the B7-H5 polypeptides or functional fragments containingtargeting signals will be generated by PCR or other standard geneticengineering or synthetic techniques.

A promoter is a TRE composed of a region of a DNA molecule, typicallywithin 100 base pairs upstream of the point at which transcriptionstarts. Enhancers provide expression specificity in terms of time,location, and level. Unlike a promoter, an enhancer can function whenlocated at variable distances from the transcription site, provided apromoter is present. An enhancer can also be located downstream of thetranscription initiation site. To bring a coding sequence under thecontrol of a promoter, it is necessary to position the translationinitiation site of the translational reading frame of the peptide orpolypeptide between one and about fifty nucleotides downstream (3′) ofthe promoter. The coding sequence of the expression vector isoperatively linked to a transcription terminating region.

Suitable expression vectors include plasmids and viral vectors such asherpes viruses, retroviruses, vaccinia viruses, attenuated vacciniaviruses, canary pox viruses, adenoviruses and adeno-associated viruses,among others.

Polynucleotides can be administered in a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers are biologicallycompatible vehicles which are suitable for administration to a human,e.g., physiological saline. A therapeutically effective amount is anamount of the polynucleotide which is capable of producing a medicallydesirable result (e.g., an enhanced T cell response) in a treatedanimal. As is well known in the medical arts, the dosage for any onepatient depends upon many factors, including the patient's size, bodysurface area, age, the particular compound to be administered, sex, timeand route of administration, general health, and other drugs beingadministered concurrently. Dosages will vary, but a preferred dosage foradministration of polynucleotide is from approximately 10⁶ to 10¹²copies of the polynucleotide molecule. This dose can be repeatedlyadministered, as needed. Routes of administration can be any of thoselisted above.

Included in these in vivo approaches, are methods of co-stimulating a Tcell that involve administering more than one co-stimulatory molecule orfunctional fragment thereof. Such combinations can be any combination ofone or more of co-stimulatory polypeptides, e.g., B7-1, B7-2, B7-H1,B7-H2, B7-H3, B7-H4, B7-H5, 4-1BB, OX40, or HVEM and functionalfragments of any of these. The proteins or functional fragments per secan be administered or nucleic acids (e.g., expression vectors) encodingthe proteins or functional fragments can be administered. Whereexpression vectors are used, a single vector containing coding sequencesfor two or more of the co-stimulatory polypeptides or functionalfragments can be administered. Alternatively, multiple (e.g., 2, 3, 4,5, or 6) individual vectors, each encoding one or more (e.g., 2, 3, 4,5, or 6) of the co-stimulatory polypeptides or functional fragmentsthereof can be administered.

Ex vivo Approaches

Peripheral blood mononuclear cells (PBMC) can be withdrawn from thepatient or a suitable donor and exposed ex vivo to an activatingstimulus (see above) and a B7-H5 polypeptide or polypeptide fragment(whether in soluble form or attached to a sold support by standardmethodologies). The PBMC containing highly activated T cells are thenintroduced into the same or a different patient.

An alternative ex vivo strategy can involve transfecting or transducingcells obtained from the subject with a polynucleotide encoding a B7-H5polypeptide or functional fragment-encoding nucleic acid sequencesdescribed above. The transfected or transduced cells are then returnedto the subject. Such cells are preferably hematological cells (e.g.,bone marrow cells, macrophages, monocytes, dendritic cells, or B cells),although they can also be any of a wide range of types including,without limitation, fibroblasts, epithelial cells, endothelial cells,keratinocytes, or muscle cells in which they act as a source of theB7-H5 polypeptide or functional fragment thereof for as long as theysurvive in the subject. The use of hematological cells that include theabove APC would be particularly advantageous in that such cells would beexpected to home to, among others, lymphoid tissue (e.g., lymph nodes orspleen) and thus the B7-H5 polypeptide or functional fragment would beproduced in high concentration at the site where they exert theireffect, i.e., enhancement of an immune response. In addition, if APC areused, the APC expressing the exogenous B7-H5 molecule can be the sameAPC that presents an alloantigen or antigenic peptide to the relevant Tcell. The B7-H5 polypeptides can be secreted by the APC or expressed onits surface. Prior to returning the recombinant APC to the patient, theycan optionally be exposed to sources of antigens or antigenic peptidesof interest, e.g., those of tumors, infectious microorganisms, orautoantigens. The same genetic constructs and trafficking sequencesdescribed for the in vivo approach can be used for this ex vivostrategy. Furthermore, tumor cells, preferably obtained from a patient,can be transfected or transformed by a vector encoding a B7-H5polypeptide or functional fragment thereof. The tumor cells, preferablytreated with an agent (e.g., ionizing irradiation) that ablates theirproliferative capacity, are then returned to the patient where, due totheir expression of the exogenous B7-H5 polypeptides (on their cellsurface or secreted), they can stimulate enhanced tumoricidal T cellimmune responses. It is understood that the tumor cells that, aftertransfection or transformation, are injected into the patient, can alsohave been originally obtained from an individual other than the patient.

The ex vivo methods include the steps of harvesting cells from asubject, culturing the cells, transducing them with an expressionvector, and maintaining the cells under conditions suitable forexpression of the B7-H5 polypeptide or functional fragment. Thesemethods are known in the art of molecular biology. The transduction stepis accomplished by any standard means used for ex vivo gene therapy,including calcium phosphate, lipofection, electroporation, viralinfection, and biolistic gene transfer. Alternatively, liposomes orpolymeric microparticles can be used. Cells that have been successfullytransduced are then selected, for example, for expression of the codingsequence or of a drug resistance gene. The cells may then be lethallyirradiated (if desired) and injected or implanted into the patient.

It is understood that in these ex vivo procedures, the cells to beintroduced into a subject can be transfected or transformed with one ormore (e.g., two, three, four, five, or six) expression vectorscontaining one or more (e.g., two, three, four, five, or six) sequencesencoding any of the co-stimulatory molecules listed above (e.g., B7-1,B7-2, B7-H1, B7-H2, B7-H3, B7-H4, or B7-H5) or functional fragmentsthereof prior to introduction.

Methods of Screening for Compounds that Inhibit or Enhance ImmuneResponses

The invention provides methods for testing compounds (small molecules ormacromolecules) that inhibit or enhance an immune response. Such amethod can involve, e.g., culturing a B7-H5 polypeptide of the invention(or a functional fragment thereof) with T cells in the presence of a Tcell stimulus (see above). The B7-H5 molecule can be in solution ormembrane bound (e.g., expressed on the surface of the T cells) and itcan be natural or recombinant. Furthermore, the B7-H5 polypeptides (orfunctional fragments thereof) can have amino acid sequences identical towild-type sequences or they can have one or more conservativesubstitutions. Compounds that inhibit the T cell response will likely becompounds that inhibit an immune response while those that enhance the Tcell response will likely be compounds that enhance an immune response.

The invention also relates to using B7-H5 or functional fragmentsthereof to screen for immunomodulatory compounds that can interact withB7-H5. One of skill in the art would know how to use standard molecularmodeling or other techniques to identify small molecules that would bindto T cell interactive sites of B7-H5. One such example is provided inBroughton (1997) Curr. Opin. Chem. Biol. 1, 392-398.

A candidate compound can modulate, e.g., inhibit or enhance, an immuneresponse. A candidate compound that causes a requirement for at least1.5-fold (e.g., 2-fold, 4-fold, 6-fold, 10-fold, 150-fold, 1000-fold,10,000-fold, or 100,000-fold) more B7-H5 in order to achieve a definedarbitrary level of T cell activation than in the absence of the compoundcan be useful for inhibiting an immune response. On the other hand, acandidate compound that causes a requirement of at least 1.5 fold (e.g.,2-fold, 4-fold, 6-fold, 10-fold, 100-fold, 1000-fold, 10,000 fold, or100,000-fold) less B7-H5 to achieve a defined arbitrary level of T cellactivation than in the absence of the compound can be useful forenhancing an immune response. Compounds capable of interfering with ormodulating B7-H5 function are good candidates for immunosuppressiveimmunoregulatory agents, e.g., to modulate an autoimmune response orsuppress allogeneic or xenogeneic graft rejection.

B7-H5 Antibodies

The invention features antibodies that bind to the B7-H5 polypeptides orfragments of such polypeptides. Such antibodies can be polyclonalantibodies present in the serum or plasma of animals (e.g., mice,rabbits, rats, guinea pigs, sheep, horses, goats, cows, or pigs) whichhave been immunized with the relevant B7-H5 polypeptide or peptidefragment using methods, and optionally adjuvants, known in the art. Suchpolyclonal antibodies can be isolated from serum or plasma by methodsknown in the art. Monoclonal antibodies that bind to the abovepolypeptides or fragments are also embodied by the invention, e.g., the5H9 or the 1H11 monoclonal antibody disclosed herein. Methods of makingand screening monoclonal antibodies are well known in the art.

Once the desired antibody-producing hybridoma has been selected andcloned, the resultant antibody can be produced by any of a number ofmethods known in the art. For example, the hybridoma can be cultured invitro in a suitable medium for a suitable length of time, followed bythe recovery of the desired antibody from the supernatant. The length oftime and medium are known or can be readily determined.

Additionally, recombinant antibodies specific for B7-H5, such aschimeric and humanized monoclonal antibodies comprising both human andnon-human portions, are within the scope of the invention. Such chimericand humanized monoclonal antibodies can be produced by recombinant DNAtechniques known in the art, for example, using methods described inRobinson et al., International Patent Publication PCT/US86/02269; Akiraet al., European Patent Application 184,187; Taniguchi, European PatentApplication 171,496; Morrison et al., European Patent Application173,494; Neuberger et al., PCT Application WO 86/01533; Cabilly et al.,U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application125,023; Better et al. (1988) Science 240, 1041-43; Liu et al. (1987) J.Immunol. 139, 3521-26; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84,214-18; Nishimura et al. (1987) Canc. Res. 47, 999-1005; Wood et al.(1985) Nature 314, 446-49; Shaw et al. (1988) J. Natl. Cancer Inst. 80,1553-59; Morrison, (1985) Science 229, 1202-07; Oi et al. (1986)BioTechniques 4, 214; Winter, U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321, 552-25; Veroeyan et al. (1988) Science 239, 1534; andBeidler et al. (1988) J. Immunol. 141, 4053-60.

Also included within the scope of the invention are antibody fragmentsand derivatives which contain at least the functional portion of theantigen binding domain of an antibody that binds specifically to B7-H5.Antibody fragments that contain the binding domain of the molecule canbe generated by known techniques. For example, such fragments include,but are not limited to: F(ab′)₂ fragments which can be produced bypepsin digestion of antibody molecules; Fab fragments which can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments; andFab fragments which can be generated by treating antibody molecules withpapain and a reducing agent. See, e.g., National Institutes of Health, 1Current Protocols In Immunology, Coligan et al., ed. □⊐2.8, 2.10 (WileyInterscience, 1991). Antibody fragments also include Fv (e.g., singlechain Fv (scFv)) fragments, i.e., antibody products in which there areno constant region amino acid residues. Such fragments can be produced,for example, as described in U.S. Pat. No. 4,642,334 which isincorporated herein by reference in its entirety.

Structure of B7-CD28 Family Molecules

All of the B7-like molecules, e.g., B7-H5, and their receptors are typeI transmembrane glycoproteins and are members of the immunoglobulin (Ig)superfamily. The members of B7 family share 20-35% identity in theiramino acid sequences. Despite such low homology in primary amino acidcomposition, these molecules share a similar secondary structure: singleIg V-and Ig C-like extracellular domain. Four cysteine residues, whichare involved in the formation of the disulfide bonds of the IgV and IgCdomains, are well conserved. The receptors for the B7 family are membersof the CD28 family, and possess a single IgV-like extracellular domain.Their cytoplasmic tails contain putative SH2- and SH3-motifs thought tobe involved in signal transduction.

By crystallography and molecular modeling, tertiary structures forligands and receptors of the B7-CD28 superfamily have been determined.The interactions of receptor-ligand pairs are mediated predominantlythrough residues in their IgV domains. In general, IgV domains aredescribed as two-layered (3-strands with “front” and “back” sheets. Thefront and back sheets of CTLA-4 IgV domain consist of strands A′GFCC'and ABEDC″, respectively, whereas the front and back sheets of theB7-1/B7-2 IgV domains are composed of strands AGFCC′C″ and BED,respectively. The binding faces between CTLA-4/CD28 and B7-1/B7-2 aredominated by the interaction of the CDR3 analogous loop fromCTLA-4/CD28, centered on the MYPPPY motif, with the surface formedpredominately by conserved residues between B7-1 and B7-2 on the G, F,C, C′ and C″ strands. The MYPPPY motif is not conserved in induciblecostimulatory molecules (ICOS), but a related FDPPPF sequence in theanalogous position is identified as a major determinant for binding ofICOS to B7-H2. Although the location of the PD-1 binding sites inB7-H1/B7-DC corresponds to the CTLA-4/CD28 binding sites in B7-1/B7-2,B7-H1 and B7-DC utilize non-conserved residues on their A′GFCC′C″ faceto bind PD-1. Crystal structures of CTLA-4/B7complexes contain bivalenthomodimers of CTLA-4 with B7-binding sites located distally to theCTLA-4 dimer interface, which suggests that the CTLA-4 homodimer canbind to noncovalent homodimers of B7-1 or B7-2 to form a lattice ofCTLA-4/B7 interactions. Formation of such a lattice is thought totrigger the formation of stable signaling complexes as part of theimmunological synapse. One of skill in the art will appreciate that thethree-dimensional structure of B7-H5 molecules is likely to be similarto that of other B7 family members.

The following example is meant to illustrate, not limit, the invention.

EXAMPLE

A human B7-H5 cDNA (FIG. 1; SEQ ID NO:5) was identified from the NCBIdatabase based on homology to other B7 family molecules, including B7-1(CD80) , B7-2 (CD86), B7-H1 (PD-L1), B7-H2 (B7h/B7RP-1), B7-H3, B7-DC(PD-L2) and B7-H4 (B7x). Full-length human B7-H5 cDNA (FIG. 9; SEQ IDNO:2) was amplified by PCR with PFU polymerase (Stratagene, CA) fromhuman placental cDNA (Clontech, CA), cloned into pcDNA™3.1⁻ vector(Invitrogen, CA) and confirmed by DNA sequencing. A mouse B7-H5 cDNA(FIG. 2, SEQ ID NO:6) was identified from the NCBI and Celera databasesbased on homology to the same B7 family molecules. To isolate the mouseB7-H5 homologue, several sets of primers based on mouse and human ESTsequences were used to amplify mouse B7-H5 cDNA (FIG. 10; SEQ ID NO:4)from spleen cDNA of a C57BL/6 (B6) mouse. Full-length mouse B7-H5 cDNAwas similarly cloned into pcDNA™3.1⁻ vector and confirmed by DNAsequencing.

Molecular models of the N-terminal V-domains of human B7-H5 (FIG. 5) andmouse B7-H5 were built by homology modeling based on the X-raystructures of human CD80 and CD86 (Stamper et al, 2001, Nature410:08-611; Schwartz et al., 2001, Nature 410:604-608) using MOE(Molecular Operating Environment, Chemical

Computing Group, Quebec, Canada). Insertions and deletions in mouse andhuman B7-H5 relative to the structural template(s) were modeledemploying a protein database segment matching procedure (Levitt, 1992,J. Mol. Biol. 226:07-533; Fechteler et al., 1995, J. Mol. Biol.253:114-131) implemented in MOE. Side chain replacements were carriedout using a rotamer library (Ponder and Pichards, 1987, J. Mol. Biol.193:775-791) extracted from high-resolution protein data bank structures(Berman et al., 2000, Nucleic Acids Res. 28:235-242). Intramolecularcontacts and stereochemistry of the models were optimized by limitedenergy minimization using protein force field parameters (Engh andHuber, 1991, Acta Crystallogr. A47:392-400). Residue mapping studies andcomputer graphical analysis were carried out with InsightII (MSI, CA).Both human and mouse B7-H5 polypeptides were predicted to contain anIgV-like domain , which is involved in the interaction of B7 moleculeswith their cognate receptors. The human and mouse B7-H5 polypeptides arealso predicted to contain a single transmembrane domain, a cysteinepredicted to be involved in dimerization, e.g., heterodimerization, anda tyrosine in the cytoplasmic domain predicted to be phosphorylatedduring signaling. Two structural cysteines, as indicated in FIG. 3, arehighly likely to contribute to the formation of the IgV domain.Additional amino acids can form a non-typical immunoglobulinconstant-like region as indicated in FIG. 5.

FIG. 6 shows an alignment of the immunoglobulin superfamily (IgSF)V-domain sequences of the B7 family (h: human, m: mouse), includingB7-H5. IgSF V-set consensus residues were defined according to Williamsand Barclay (1998, Annu. Rev. Immunol. 6:381-405) and Bork et al (1994,J. Mol. Biol. 242:309-320), and are shown on a black background.Consensus residue positions are labeled with invariant IgSF residues orconserved residue character (h: hydrophobic, p: polar). Residuepositions shown on a gray background are signature residues of the B7family outside IgSF consensus positions. Beta-strands of the V-domainare designated according to IgSF conventions (A′, B, C, C′, C″, D, F,G). Residue positions labeled with # are involved in dimerization ofCD80/CD86 in their crystal structures and residues labeled withasterisks participate in cytotoxic T-lymphocyte-associated protein 4(CTLA4) binding.

On the basis of this alignment, in both human and mouse B7-H5, 14/16IgSF V-set consensus residues and 12/13 B7 signature residues areconserved. This is strong evidence that these proteins have V-like B7domains.

The most unique features in these domains are unusual insertions in theA′-B and C-D loops, including free cysteines. These loops are spatiallyadjacent in the C-terminal region of the V-domain, at its interface withsubsequent regions. The cysteine residues would be available forinter-loop disulfide bonding or, alternatively, covalent interactionswith adjacent domains. Given these insertions, the C-terminal region ofthe B7-H5 V-domain would likely not be capable of forming the interfacewith a subsequent C-type IgSF domain, as seen in the crystal structureof CD80.

Fusion protein constructs of human B7-H5 polypeptide (FIG. 3; SEQ IDNO:1) and mouse B7-H5 polypeptide (FIG. 4; SEQ ID NO:3) were prepared bycloning the extracellular domain of B7-H5 in frame with thehinge-CH2-CH3 domain of either human IgG1 or mouse IgG2a (Chapoval etal., 2002, Mol. Biotechnol. 21:259-264). To enhance the secretion offusion protein, the native signal peptide of B7-H5 was replaced with thepreprotrypsin signal peptide and FLAG sequence derived from pCMV-FLAG™vector (Sigma, MO). To produce the B7-H5Ig fusion proteins, 293T cellswere transfected with 10 pg of mouse or human B7-H5Ig constructs by thecalcium phosphate method, and B7-H5Igs were purified from culturesupernatant by protein G column, as described previously (Dong et al.,1999, Nature Med. 5:1365-1369). Stable CHO cell lines expressing humanB7-H5 or mock-transfected lines were prepared by cotransfection ofpcDNA™ vector containing human B7-H5 cDNA with pLXSHD, a plasmidencoding histidinol-resistant gene (Miller et al., 1993, MethodsEnzymol. 217:581-599). Stable clones were selected with 20 mM histidinol(Sigma, MO). Clones expressing B7-H5 were screened for binding withhuman B7-H5 monoclonal antibody (mAb).

Monoclonal antibodies to human B7-H5 were generated by immunization of aBALB/c mouse by immunization methods described previously (Wilcox etal., 2002, J. Clin. Invest. 109:651-659). Two hybridomas, 5H9 and 1H11,which secrete mouse IgG1 against human B7-H5 (see FIGS. 8A and 8B) weregenerated. The monoclonal antibodies produced by the two hybridomas werepurified by IgG affinity column chromatography. Specificity of the mAbwas determined by negative staining of various transfectants expressingB7 family molecules including B7-1, B7-2, B7-H1, B7-DC, B7-H2 and B7-H3.Control mouse IgG1 were purchased from Rockland (Gilbertville, Pa.).

The activity of human B7-H5 to stimulate proliferation of T cells wasinvestigated. Flat bottom 96-well plates were coated with varyingconcentrations of anti-CD3 mAb (Dong et al, 1999, Nature Med.5:1365-1369), washed extensively, and coated with 10 μg/ml B7-H51g orcontrol mIg for 2 hours at 37° C. Nylon wool-purified human CD3+ T cellsfrom peripheral blood mononuclear cells (PBMC) of healthy donors werecultured in the presence of the pre-coated anti-CD3 mAb at 3×10⁵cells/well. Seventy-two hours later, the wells were pulsed with 1 μCi of³H-thymidine (TdR) and the proliferation of T cells was determined byincorporation of TdR. The B7-H5Ig fusion protein stimulated T cellproliferation at concentrations as low as 0.6 μg/ml (FIG. 7). Thisdemonstrates that the B7-H5 polypeptide can co-stimulate T cells.

What we claim is:
 1. An isolated nucleic acid comprising: (a) a nucleicacid sequence that (i) encodes a B7-H5 polypeptide with the ability toco-stimulate a T cell, and (ii) hybridizes under highly stringentconditions to the complement of SEQ ID NO:2 or SEQ ID NO:4; or (b) thecomplement of the nucleic acid sequence in (a).